Performance evaluation of a hyphenated laser spectroscopy system with conventional methods for microplastic analysis

Microplastics are one of the concerning environmental pollutants because of their ubiquity. Their capability to adsorb other environmental pollutants increases the risk even further. Existing identification approaches for microplastic characterization for polymer class and their surface-adsorbed heavy metal detection require the utilization of multiple resources and expertise. The article discusses the applicability of a custom-made hyphenated Laser Induced Breakdown Spectroscopy (LIBS)—Raman spectroscopic system in characterizing microplastics by comparing the analytical performance with conventional methods such as Attenuated Total Reflectance- Fourier Transform Infrared (ATR-FTIR) spectroscopy, confocal Raman spectroscopy, and Scanning Electron Microscopy–Energy Dispersive X-ray Spectroscopy (SEM–EDS). Raman analysis identified polyethylene (PE), polypropylene (PP), and polyethylene terephthalate (PET) plastics, which is confirmed by confocal Raman and FTIR study of the same. LIBS study of microplastics detected heavy metals such as Al, Ni, Co, and Zn, along with Ca and Mg trace elements. The cross-examination with EDS validates these trace elements' presence on the microplastics' surface. The results of the reported LIBS-Raman analysis and its validity evaluated using conventional gold-standard methods show the applicability of the proposed methodology in characterizing microplastics from environmental resources with less or no sample preparation in short time.

Plastics are synthetic polymers consisting of long chains of monomers, which have become an essential part of our lives due to their durability 1 .However, the mismanagement of plastic waste is causing some concern because of its threat to humans and the environment [2][3][4] .This threat of solid plastic waste accumulation has multiplied in the past decade, and it is reflected in the abundance of microscopic plastic particles (microplastics) in the environment 5 .Microplastics refer to plastic particles having a dimension between 1 µm and 5 mm [6][7][8] .They are either produced in these dimensions for industrial applications, termed primary microplastics or are resulted from the degradation of larger plastics, termed secondary microplastics.Due to their increased specific surface area compared to the solid pieces, the adsorption of environmental pollutants like Persistent Organic Pollutants (POPs), bio-film, and heavy metals takes place 9 .
Heavy metals are metallic elements with a density 10 greater than 5 g/cm 3 .They are considered hazardous environmental pollutants among these contaminants since they can form lipophilic ions/compounds when they form bonds with non-metallic molecules inside the living cell and can lead to cellular toxicity 11 .Anthropogenic activities like mining, usage of pesticides, and industrial wastewater introduce heavy metals into the environment 12 .
Due to the weathering of plastic fragments, the specific surface area (ratio of surface area to mass) increases with enhancement in surface roughness that aids in the adsorption of heavy metal contaminants onto the surface 13 .The adsorption rate of heavy metals among different classes of plastics itself varies and adds complexity further 14 .Some plastics also contain heavy metallic additives in their polymer matrix, which are added to attain the desired characteristics, and once waste plastics reach the environment, they can leach out from the polymer matrix 15 .Chen et al. 16 , with the help of an in-vitro digestion model, demonstrated the desorption of adsorbed heavy metal contaminants from the microplastics subjected to artificial gastric and intestinal juice, which is an example of the vector effect of microplastics in the translocation of heavy metals into the human body.Further, the potentiating effect of the adsorption and desorption of heavy metals with microplastics needs to be explored to assess their ill effects.
The necessity of applying multiple analytical methods for microplastics and their surface-adsorbed heavy metal detection hinders rapid characterization and demands the use of several harsh chemicals for multistep sample preparation and analysis as well.These methods also demand the usage of multiple resources.To address this barrier of the multistep analysis process, the integration of complementary techniques under a single analytical system that shares a similar instrumentation design has proven to be practical for different analytical problems 31 .One such system is a bimodal LIBS-Raman spectroscopy, which combines elemental and molecular identification 32 .The combination of LIBS and Raman spectroscopy under a single system has several advantages over other techniques, such as cost-effectiveness, minimal sample preprocessing, and rapid analysis 33 .The first-hand results on the microplastic-heavy metal detection using the LIBS-Raman system can be found in our earlier publication 30 .
In this article, we examine, in detail, the potential of the LIBS-Raman spectroscopy system in characterizing microplastics from environmental samples by comparing the results with conventional identification techniques and validating the characterization performance.We utilize Raman spectroscopy to characterize microplastics and LIBS to diagnose surface-adsorbed heavy metals.The system's ability to completely characterize microplastics using the proposed system is demonstrated by comparing the results with existing gold standards like ATR-FTIR, confocal Raman spectroscopy, and SEM-EDS.The developed methodology requires a simple sample extraction and pre-treatment steps, which helps the rapid analysis.
In comparison with our earlier publication 32 , this article discusses the validation of a LIBS-Raman spectroscopic system in identifying the polymer class as well as the adsorbed heavy metal contaminants on microplastics in the size range of 1-5 mm with conventional methods.A total of six microplastic samples collected from an estuary were characterized by the LIBS-Raman spectroscopic system and the commercial systems, namely, ATR-FTIR and SEM coupled with EDS, and the results were evaluated to illustrate the competence of the system over traditional techniques requiring multiple systems for complete analysis.The comparison of results obtained by the lab-built LIBS-Raman system is at par with conventional methods.

Methodology Collection and extraction of microplastics
For the current study, the microplastic samples were collected from an estuary on the west coast of India, formed by the River Netravathi in Mangalore, Karnataka, India.From the sampling site, 100 L of water was collected using a 10 L stainless-steel bucket and subjected to sieving with the help of a stack of stainless-steel sieves with pore sizes 5 mm and 1 mm.Before collecting the River water, all equipment was rinsed with distilled water, followed by river water.Then, water was collected using the stainless-steel bucket and poured into the stack of sieves.The macro debris (> 5 mm) that remained on the top of the sieve (with 5 mm pore size) was discarded, and the residues collected in the sieve (with 1 mm pore size) were transferred to a stainless-steel container with the help of distilled water, The sample was subjected to preprocessing at the laboratory using the extraction protocol by Masura et al. 34 and Amrutha et al. 35 .
In brief, the samples were subjected to wet sieving with stacked stainless-steel sieves with 5 mm and 1 mm pore sizes.The residue was transferred to a glass beaker and kept in a hot air oven for a duration of 24 h at 50 °C.Then, the samples were subjected to Wet Peroxide Oxidation (WPO) to digest the organic matter with the help of 20 mL of 30% v/v hydrogen peroxide (H 2 O 2 ).Then, 20 mL of 0.05 M ferrous solution was added to the beaker and heated using a hot plate.Hydrogen peroxide was added drop by drop till all the organic material was dissolved.This procedure is necessary to remove the organic contaminants residing on the microplastics' surface, which will aid in extracting microplastics from the sample by adopting the density separation method.Although, the WPO with hydrogen peroxide will change the valency of the heavy metal contaminants when the concentration of H 2 O 2 used is effectively high 36 , in our study, we used 20 mL of 30% H 2 O 2 solution, which is only sufficient to digest the organic contaminants and has minimal effect on the change in the valence state of the metal ions leading to the desorption.Also, from our early reports, it is clear that the WPO process with the specified concentration of hydrogen peroxide dissolves only the organic surface contaminants and does not induce any chemical or structural changes to the microplastics within 24 h, which is evident from Raman spectral analysis of microplastics with and without WPO 30 .For the selection of suitable salt for extracting the microplastics, a literature survey was conducted, and the results are as follows.Several studies have been conducted with different salt solutions, including NaCl, NaBr, and ZnCl 2, on the aspect of microplastic recovery from different environmental samples like sediment, water, soil, etc.The recovery rate observed by Radford et al. 37 in extracting microplastics from soil samples with NaCl solution (1.2 g/cm 3 density) was around 60%, whereas Vermeiren et al. 38 achieved > 90% recovery using ZnCl 2 solution (1.7 g/cm 3 density) [37][38][39] .Hence, ZnCl 2 was chosen for density separation, which has a recovery rate of > 90%.In order to increase the density of the aqueous solution, ZnCl 2 solution was added to the beaker (concentration 933.3 g/L).Then, the mixture was transferred to a density separator and kept aside to allow the denser organic materials to settle down.Since the density of polymers, like 1.3 g/cm 3 for PVC and 1.45 g/cm 3 for PET, is lesser than the density of the ZnCl 2 solution (1.7 g/cm 3 ) used in our study, the recovery rate will be maximum.The presence of small amounts of heavy metal contaminants does not significantly change the density of these microplastics; hence, to the best of our understanding, their recovery rate will not be affected.Following this, the organic matter that got settled down was drained off, and the microplastics floating in the supernatant were sieved once again with 5 mm and 1 mm sieve, transferred to a glass container, and dried.
A total of six microplastics were extracted and were named MPS1, MPS2, MPS3, MPS4, MPS5, and MPS6.The characterization of the extracted samples was done based on their morphology, polymer class, and adsorbed heavy metals.

Visual identification
The microplastic samples were transferred into a watch glass and visualized under the Nikon Eclipse Ni microscope (40× and 100× magnification) to analyze their morphology.The brightfield images were captured with resolutions of 2.12 μm and 0.92 μm with 4× and 10× objective lenses.Then, they were categorized into fragments, films, and fibers based on shape and size 34 .

Identification of polymer class
A locally developed LIBS-Raman spectroscopic system was used to characterize the microplastics, and a detailed description of the system is discussed in our previous article 32 .In brief, a frequency-doubled, Q-switched Nd:YAG laser (Quantel Q-smart 450) was used as an excitation source.The laser operates at 532 nm with a 6 ns pulse width and 10 Hz repetition rate.The LIBS or Raman signals are collected using a back-collection optical configuration.
The Czerny-Turner spectrograph (Kymera 328i, Andor) with multiple grating options (600 lines/mm, 1200 lines/mm blazed at 500 nm, and a holographic grating 2400 lines/mm blazed at 300 nm) is used for Raman analysis.In the current study, 600 lines/mm grating with ~ 130 nm bandpass is used for the spectroscopic analysis.The spectra were recorded using a CCD detector (iDus -420, Andor) operating with the synchronized trigger from the laser.
The LIBS spectral analysis was carried out by coupling the atomic emission signals from the sample to the Echelle spectrograph (Mechelle ME5000, Andor) with an ICCD detector (iStar, Andor) using an optical fiber cable.The spectrograph has a spectral coverage of around 775 nm in a single scan with a high resolution of 0.01 nm.The ICCD was synchronized with the laser source via the Digital Storage Oscilloscope (DSO) for gate monitoring.The LIBS spectra were recorded in single-shot mode, with optimized gate width and gate delay as 10 μs and 700 ns, respectively.

Microplastics analysis using Raman spectroscopy
The polymer class of the collected microplastics was identified using Raman spectroscopy with the help of the LIBS-Raman system.The microplastic samples were sandwiched between two glass slides as prescribed in a study 30 .The extended part of the microplastics out of the slides was introduced at the focal point of the focusing lens.The energy of the laser beam was reduced to 0.8 mJ using the energy regulator, and with external triggering from the laser, the spectra were recorded with an exposure time of 5 s in the spectral range 278-3760 cm −1 with a resolution of 2.76 cm −1 .The collected Raman spectra were baselined using the asymmetric least square method with OriginPro software.The polymer class of each microplastic was identified by comparing the Raman spectra of standard polymer samples and with the literature [40][41][42][43][44][45] .
Further, the samples were also analyzed under a commercial Raman microscope (XpolRA PLUS, HORIBA Scientific) with a 532 nm laser source, 20× objective lens, and 1800 lines/mm grating to compare the lab-based system's ability.The Raman spectra were acquired with 2 s acquisition time.The obtained Raman spectra were smoothened using the Savitzky-Golay method with a window size of 10 points and baselined using the asymmetric least square method with OriginPro (OriginPro 2022 (9.9) SR1, OriginLab, https:// www.origi nlab.com/ index.aspx?go= Produ cts/ Origin/ 2022& pid= 4418) software.

LIBS analysis of microplastics
The microplastic samples were subjected to LIBS analysis to identify the surface-adsorbed heavy metals.The laser energy was increased to 4 mJ, and the gate delay was set to 700 ns with a gate width of 10 μs.The LIBS spectra acquired were analyzed for adsorbed contaminants referring to the NIST (National Institute of Standards and Technology) atomic spectra database.www.nature.com/scientificreports/

SEM-EDS analysis of microplastics
The cross-verification of the LIBS results on adsorbed heavy metal contaminants was performed along with the determination of the extent of degradation of microplastics, using the gold-standard methods-Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS).The microplastics were subjected to sputter coating with gold to develop a thin film of gold on the surface of the microplastics, and the SEM analysis was carried out by fixing the samples onto a stainless steel substrate and analyzed using Hitachi S-3400, operating at 15 kV.The elemental mapping of the microplastics with EDS characterization was carried out using a ThermoNORAN NSS detecting system.

Visual identification and characterization of microplastics
The brightfield images of the six microplastics are shown in Fig. 1.Out of six microplastics, two are identified as fibers, two as fragments, and the remaining two as films.The sources of fibrous microplastics and films can be attributed to the discharge of fibers from synthetic cloths and single-use carry bags 35 .Based on the microplastics' morphological characteristics, all are identified as secondary microplastics because of their roughed and degraded surfaces 50 .

SEM analysis of microplastics
The SEM images of the microplastics are shown in Fig. 2.After conducting all other characterizations mentioned in the methodology section, the SEM study was carried out as the conductive coating of gold is necessary for SEM characterization.The SEM images of the six microplastics revealed their highly degraded surface.Sample MPS1 had an uneven, roughened surface with craters and pits caused by weathering 51 .Sample MPS2 had a flaky surface, possibly caused by aberrant degradation 50 .Sample MPS3 was fibrous, and some microfractures were observed with smooth surfaces.Sample MPS4 had the least amount of surface degradation among the collected microplastics, with only a few minor fractures and cracks.Sample MPS5 was also fibrous, with a comparatively rougher surface than MPS3, and some flakes were embedded between the fibers.Sample MPS6 had a high degree of degradation, with protrusions, cracks, and a flaky surface.

Raman spectroscopic characterization
The Raman spectral comparison of extracted microplastics is shown in Fig. 3. Sample MPS1 and MPS2 were identified as polyethylene with two eminent peaks at 1292 cm −1 and 1445 cm −1 , which were designated to CH 2 twisting CH 2 symmetric deformation.Two more peaks in the region 2880 cm −1 and 2845 cm −1 corresponding to CH 2 asymmetric and symmetric stretching vibration were also observed.Apart from the characteristic peaks, a peak at 1716 cm −1 was observed in the Raman spectra of sample MPS1.It was assigned to C=O stretching vibration, which might be due to weathering-induced oxidation 30 .Sample MPS3 and MPS5 were identified as polyethylene terephthalate with characteristic peaks at 1609 cm −1 and 1718 cm www.nature.com/scientificreports/stretching vibrations respectively.Apart from those three peaks, three more peaks at 1456 cm −1 , 1325 cm −1 , and 1147 cm −1 were also observed, which were assigned to CH 3 asymmetric bending, C-H symmetric bending, and C-C stretching vibrations.Cross-verifying the polymer class of the microplastic samples was carried out under a commercial Raman microscope (XpolRA PLUS, HORIBA Scientific) with a 20× objective lens.The Raman spectra were acquired using LabSpec 6 software with 2 s acquisition time and 200 µm slit width.A comparison of the normalized spectra obtained using the lab-based and commercial systems is shown in Fig. 3.The spectral signatures observed by both systems are similar, and the spectral assignments are tabulated in Table 1.Thus, we can conclude that our system can efficiently distinguish between different classes of plastics.

ATR-FTIR analysis
The ATR-FTIR analysis of the collected microplastics using the commercial system was carried out to further cross-verify the results obtained from the system based on Raman spectroscopy as they are complementary to each other in terms of molecular structure-based analysis.The FTIR spectra were collected by placing the microplastics onto the ATR crystal made up of diamond, and the spectra were analyzed in the wavenumber range of 600-4000 cm −1 .The FTIR spectra of three samples, MPS2, MPS3, and MPS5, are shown in Fig. 4. The spectral assignments corresponding to the microplastic samples are tabulated in Table 2.  www.nature.com/scientificreports/ The noise level of the FTIR spectrum of MPS1 was very high, and thus, we were not able to identify the polymer class.The FTIR spectrum of MPS2 resembled the FTIR spectrum of polyethylene.The peak in the region 1378 cm −1 was due to the wagging deformation of the CH 2 .The peak in the region 1466 cm −1 was due to the CH 2 scissoring.The peaks at 2848 cm −1 and 2918 cm −1 were assigned to the symmetric and asymmetric stretching of CH 2 .Interestingly, the additional peak at 1018 cm −1 was observed, which might be due to the presence of ester groups due to the photo oxidation of MPs.The FTIR spectra of MPS4 and MPS6 resembled the FTIR spectrum of polypropylene.The peak in the region 1370 cm −1 was assigned to the symmetric bending of CH 3 .The peak at 1450-1456 cm −1 was due to the symmetric bending of CH 2 .The peaks in the region 2830-2852 and 2916-2944 cm −1 were assigned to the symmetric and asymmetric stretching of the CH 2 group.Two additional peaks between 996-1010 cm −1 and 1700-1720 cm −1 were observed, which might be due to the presence of ester and carboxyl groups due to the photo oxidation of MPs.The FTIR spectra of MPS3 and MPS5 resembled the FTIR spectrum of polyethylene terephthalate.The peaks in the spectral range 722-732 cm −1 and 872 cm −1 were due to the out-of-plane wagging and bending of aromatic C-H.The peaks in the region 1100-1102 cm −1 and 1224-1240 cm −1 were due to the C-O-C and C-C-O stretching vibrations.The peak in the region 1406-1418 cm −1 was assigned to the vibration of the phenyl ring.The peak at 1722 cm −1 was assigned to the C=O stretching of the carboxyl group.Two peaks corresponding to in-plane and out-of-plane vibration of the benzene group were identified at 1017 cm −1 and 868 cm −1 , respectively.Interestingly, the additional peak at 1022-1024 cm −1 was observed, which might be due to the presence of ester groups due to the photo oxidation of MPs.www.nature.com/scientificreports/

LIBS analysis of microplastics
The LIBS spectra were recorded in the 200-900 nm spectral range with 10 ms exposure time and analyzed in the 300-450 nm spectral range to identify the heavy metals adsorbed onto the MPs.The spectral range was selected based on the presence of intense emission lines from heavy metals that were adsorbed onto MP's surface.The spectral assignments were done by comparing the emission lines with the NIST database.LIBS spectra of all six samples are shown in Fig. 5. Elements detected from all six samples with corresponding emission lines are tabulated in Table 3.In summary, Al, Ca, and Mg were found in all six samples.Five samples, namely, MPS2, MPS3, MPS4, MPS5, and MPS6, showed the presence of Co with characteristic emission lines.Emission lines corresponding to Ni were found in five samples, namely, MPS1, MPS2, MPS3, MPS5, and MPS6.Zn was found in MPS1, MPS2, MPS4, MPS5, MPS6.

EDS analysis of microplastics
Energy-dispersive X-ray spectroscopy (EDS) was employed using SEM coupled with an EDS system to crossvalidate the presence of the elements identified by the LIBS.The summary of the results is tabulated in Table 4. Two sample EDS spectra of sample MPS2 and MPS6 are shown in Fig. 6.Most of the elements found in the LIBS analysis were also found in the EDS analysis.The major difference found was the presence of Pb, Si, and Cl in all the samples, which was not found in the LIBS analysis and might be because of the system's limit of detection.Apart from the above, Al was only found in samples MPS2 and MPS6, Mg was found only in MPS6, and Ca was only found in MPS4.
The results clearly indicate the advantage of the proposed LIBS-Raman system in completely characterizing microplastics based on chemical composition and surface adsorbed heavy metals, and proven to be accurate based on the comparison with the results of conventional gold standard methods.The existing reports on microplastic analysis discuss these aspects of characterization via completely different sample preparation and analytical approaches, whereas the current results do not require additional complexities in the process [52][53][54][55][56] .www.nature.com/scientificreports/An aspect of improving the reported system is incorporating a microscopic system in the configuration so that the characterization based on morphology can also be performed using the single system, and the analyzable size limit for the micro-particles can be brought further down.A comparison of ablation craters formed on the surface of a polymer material (polypropylene) when using the current focusing optics and microscope objectives of different magnifications are compared in Fig. 7, which clearly shows how the analyzable size can be brought down in the future.

Conclusion
In summary, the microplastics collected from River Netravathi with multiple analytical systems have been characterized.Brightfield imaging and SEM characterization of microplastics were performed to characterize extracted microplastics based on the surface morphology.The roughed cracked surface with protrusions indicates the extinction of weathering.The microplastics with a size range of 1-5 mm were successfully characterized with the custom-built Raman and LIBS instrument, which reduces the utilization of multiple resources for the complete analysis of the sample.Out of six microplastics, two were identified as polyethylene (PE), two were identified as polypropylene (PP), and the remaining two were identified as polyethylene terephthalate (PET).The system's accuracy in identifying polymer class was cross-validated with confocal Raman spectroscopy and ATR-FTIR spectroscopy, and the results agree with characteristic bands of the corresponding polymer classes.LIBS analysis of microplastics detected the heavy metals, namely, Al, Ni, Co, and Zn, along with trace elements like Ca and Mg and EDS confirms these observations, in turn, proves the applicability of the developed technique for the identification of adsorbed heavy metals in microplastics.From the observed results, the system's ability to identify MPs and surface-adsorbed heavy metals significantly helps the microplastic research advance with rapid, complete characterization.

Figure 1 .
Figure 1.Brightfield images of the collected microplastics with naming.

Figure 2 .
Figure 2. SEM images of the six microplastics.

Figure 3 .
Figure 3. Raman spectra of the microplastics acquired using lab-built and commercial confocal Raman setup with peak assignments (A) Raman spectra of PE microplastics, (B) Raman spectra of PP microplastics, (C) Raman spectra of PET microplastics.

Figure 6 .
Figure 6.EDS spectra of two microplastics (MPS2 and MPS6) showing the presence of different surface adsorbed elements including the heavy metals.

Table 1 .
Spectral assignment corresponding to Raman spectra of microplastics.

Table 2 .
Spectral assignment corresponding to FTIR spectra of microplastics.

Table 3 .
Elements detected by LIBS analysis (excluding C, O, and N).

Table 4 .
Elements detected by EDS analysis (excluding C and O).